ATP-based Luminescence in the Absence of Cytokines Measured in Cell-Based System Using Plate Reader - 2061-06_Inhibitor_Dose_DryPowder_Activity

This is a counter-screen to test compounds for ATP-based luminescence in the absence of cytokines. Cells will be treated with compounds at various doses for 48 hours. The cells will not be treated with cytokines. While perhaps interesting in another context, compounds that elevate ATP levels in INS-1E cells under basal conditions are not suitable as probes in this assay. Further, compounds that suppress cytokine-induced beta-cell apoptosis but are toxic in their own right are similarly unattractive. ..more

Validation of the affinity reagent probe (ML-187) with a PEG linker: comparison of ML-187-PEG and ML-187 cell-based efficacy Measured in Cell-Based System Using Plate Reader - 2061-11_Inhibitor_Dose_DryPowder_Activity

Assay Overview:This is a counter-screen to test compounds for ATP-based luminescence in the absence of cytokines. Cells will be treated with compounds at various doses for 48 hours. The cells will not be treated with cytokines. While perhaps interesting in another context, compounds that elevate ATP levels in INS-1E cells under basal conditions are not suitable as probes in this assay. Further, compounds that suppress cytokine-induced beta-cell apoptosis but are toxic in their own right are similarly unattractive.

PRESENCE OF CONTROLS: Neutral control wells (NC) were included on every plate.EXPECTED OUTCOME: Active compounds result in decreasing readout signal.ACTIVE CONCENTRATION LIMIT:For each sample, the highest valid tested concentration (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).NORMALIZATION:The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.A normalized activity value of 100 is defined as (2)(NC).A normalized activity value of -50 is defined as (0.5)(NC).Experimental wells values were scaled to this range.PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION(AC): AC50AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).AC values were calculated up to the active concentration limit described for each sample.PUBCHEM_ACTIVITY_OUTCOME:Activity_Outcome = 1 (inactive) when:a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, orb) AC > AC_limit, orc) compound shows activity but in a direction opposite to the expected outcomein these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to nullActivity_Outcome = 2 (active) when:AC <= AC_limitActivity_Outcome = 3 (inconclusive) when:a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, orb) The fit was not valid due to poor fit quality.PUBCHEM_ACTIVITY_SCORE:PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)Scores relate to AC in this manner:120 = 1 pM90 = 1 nM60 = 1 uM30 = 1 mM0 = 1 MPUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.Note:The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.